|Year : 2017 | Volume
| Issue : 1 | Page : 221-226
Evaluation of lymph node harvest using intra-arterial methylene blue injection into ex-vivo colorectal cancer specimens
Olfat I Elsebai1, Eman A Ahmed2, Mohamed S Ammar1, Mahmoud M. H. Khalil MBBCh 3
1 Department of General Surgery, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
3 Damanhur Oncology Center, Damanhur, Beheira Governorate, Egypt
|Date of Submission||28-Oct-2016|
|Date of Acceptance||02-Jan-2017|
|Date of Web Publication||25-Jul-2017|
Mahmoud M. H. Khalil
Damanhur Oncology Center, Damanhur, Beheira Governorate, 22511
Source of Support: None, Conflict of Interest: None
The aim of this work was to evaluate the effect of ex-vivo methylene blue injection into the feeding artery (or arteries) of the specimen removed for colorectal cancer to improve lymph node harvest and staging through a randomized controlled trial.
TNM stage of colorectal cancer with exact nodal staging correlates with survival rates and prognosis estimation and it is the foundation on which all treatment regimens are based.
Patients and methods
This prospective, clinical, randomized study included 50 patients suffering from colorectal cancer. They were divided into two groups: group I (the study group), in which surgically resected specimens were subjected to ex-vivo methylene blue injection into the feeding mesocolic artery, followed by pathological assessment, and group II (the control group), in which the routine conventional pathological assessment was carried out.
As regards the total number of harvested lymph nodes, the number of positive metastatic lymph nodes, and the postoperative pathological staging, there was a statistically significant difference between the two groups. There were no statistically significant differences between the two groups with respect to demographic data, tumor anatomical site, histological criteria, preoperative staging, and type of operation.
Ex-vivo methylene blue injection into the feeding mesocolic artery is a simple, easy, and safe method that significantly improves lymph node harvesting in colorectal cancer leading to a more accurate colorectal cancer staging.
Keywords: colorectal cancer, lymph node harvest, methylene blue injection
|How to cite this article:|
Elsebai OI, Ahmed EA, Ammar MS, Khalil MM. Evaluation of lymph node harvest using intra-arterial methylene blue injection into ex-vivo colorectal cancer specimens. Menoufia Med J 2017;30:221-6
|How to cite this URL:|
Elsebai OI, Ahmed EA, Ammar MS, Khalil MM. Evaluation of lymph node harvest using intra-arterial methylene blue injection into ex-vivo colorectal cancer specimens. Menoufia Med J [serial online] 2017 [cited 2020 Feb 22];30:221-6. Available from: http://www.mmj.eg.net/text.asp?2017/30/1/221/211521
| Introduction|| |
Colorectal adenocarcinoma is the second leading cause of cancer-related deaths in western countries. In Egypt, colorectal cancer contributes to 6.5% of all types of cancer .
Worldwide, many efforts have been made to find biomarkers to predict the behavior of colorectal cancer. Epithelial–mesenchymal transition is one of the central mechanisms that induces invasion and metastasis of tumors .
Overall, 90–95% of colon tumors are adenocarcinomas; the less-frequent types include carcinoids, stromal tumors (formerly leiomyosarcomas), lymphomas, and undetermined lesions. Surgery remains the cornerstone of curative therapy, with the greatest technical advances made in recent years using minimally invasive methods, including laparoscopy and robotics .
Regardless of the type of surgical approach to colon cancer, location continues to be the major determinant of the type and extent of colon resection; the degree of resection is based on the arterial, venous, and lymphatic drainage of the affected colon segment. Furthermore, medical societies and healthcare payers are increasingly relying on the adequacy of lymph node resection, and therefore the number of nodes examined histologically, as a benchmark of satisfactory oncologic therapy .
TNM stage of colorectal cancer correlates with survival rates and prognosis and it is the foundation on which all treatment regimens are based .
According to the recent guidelines for colorectal cancer management, a minimum number of 12 lymph nodes should be examined in all colorectal cancer surgical specimens to maximize the identification of regional lymph node metastasis .
It has been reported that in 75% of all colorectal specimens adequate numbers of lymph nodes are not found or examined. Furthermore, lymph nodes may harbor undetected micrometastasis, or affected small-sized lymph nodes may not be identified through routine pathologic assessment .
Manual palpation of the surgical specimen is the standard technique used by histopathologists. However, this may miss smaller nodes , and it is known that nodes smaller than 5 mm in diameter may account for up to half of the metastatic lymph nodes that are present .
To improve the lymph node harvest and reduce the non-patient-related variability, specimen preparation techniques such as fat clearance with xylene and alcohol or acetone compression have shown a significant improvement in the number of lymph nodes harvested, but fat clearance is time consuming, expensive, and involves potentially hazardous substances.
Moreover, to facilitate accurate staging, several methods of lymph node assessment have been developed. Among these methods is sentinel lymph node dissection. However, studies on sentinel lymph node sampling of the colorectal region have failed to prove the efficacy of this technique in avoiding the examination of all lymph nodes in cases of node-negative disease. These methods also include amplification using PCR and immunohistochemical analysis to detect micrometastasis. To date, none of these methods has been accepted for widespread use .
Recently, a simple ex-vivo injection of methylene blue solution into the feeding artery or arteries of the specimen has been described, resulting in significantly better lymph node detection in rectal  and colon cancers  compared with manual palpation.
| Patients and Methods|| |
This prospective, clinical, randomized study included 50 patients suffering from colorectal cancer. Their ages ranged from 20 to 70 years. All patients attended to the Department of Surgery on Menoufia University Hospital and Damanhour Oncology Center during the period of the study and research from April 2015 to March 2016. Patients who underwent elective surgery for resectable colorectal cancer with intent to cure were included. However, patients who underwent emergency surgery or palliative surgery, patients with recurrent colorectal carcinoma, and patients who received radiotherapy were excluded.
According to the review by the internal research board, because of the ex-vivo nature of the study intervention, no specific ethical approval or individual patient consent is required. All patients underwent a standard colorectal resection either by means of laparoscopic or open surgery, following discussion by multidisciplinary colorectal team. All drugs used in the research are approved by the Egyptian Ministry of Health. All patients who were included in this study were subjected to the following:
Preoperatively, the patients were subjected to the following: meticulous history taking with special emphasis on personal data, history of the present illness, medical history, previous operations, and family history; thorough clinical examination including general examination, abdominal examination, and digital rectal examination; and recording of all available data from the investigations carried out to the patients preoperatively, including laboratory investigations, tumor markers, endorectal ultrasonography, computed tomography, MRI, colonoscopy, and biopsy.
Patients with colonic malignant tumors were subjected to tumor-specific mesocolon excision. Total mesorectal excision was performed for patients with middle and lower rectal malignant tumors, whereas patients with upper rectal tumors were subjected to tumor-specific mesorectal excision. Following completion of surgical resection, all specimens in the intervention group were laid out on a table and the main feeding artery (or arteries) was carefully dissected at its highest point of ligation.
The feeding artery (or arteries) of the specimens for the stained group had been cannulated outside human body after complete resection (ex vivo) with a standard 16–20 G intravenous catheter using only the plastic tube portion, followed by the injection of 15–20 ml of methylene blue solution (50 mg methylene blue, diluted in 30 ml of sodium chloride 0.9%). Subsequently, all specimens were fixed in 10% buffered formaline for 24 h. No additional fat clearance methods were used. All specimens were sent to the Pathology Department and lymph nodes were confirmed only when a capsule was detected histologically in the stained group or the unstained group. Beginning from the proximal end, the specimens were cut into 5–7-mm-thick slices until the end of the tumor region was reached, and representative areas were embedded using the whole mount technique. The fat of the remaining parts was dissected and examined by means of palpation. Thereafter, the fatty tissue was sliced and stretched to create thin, transparent layers. The cut surfaces were screened for lymph nodes. After paraffin embedding, 3 μm sections were prepared and stained with hematoxylin and eosin. The slides were examined for the number and size of lymph nodes and the presence of metastasis.
Data were fed into the computer and analyzed using IBM SPSS software package (IBM Corporation, Armonk, New York, United States), version 20.0. Qualitative data were described using number and percentage. Quantitative data were described using range (minimum and maximum), mean, SD, and median. Significance of the obtained results was judged at the 5% level.
The following tests were used.
- χ2-Test was used for categorical variables, to compare between different groups
- Fisher's exact or Monte Carlo correction was used for correction for χ2 when more than 20% of the cells have expected count less than 5
- Student's t-test was used for normally quantitative variables, to compare between two studied groups
- Mann–Whitney test was used for abnormally quantitative variables, to compare between two studied groups.
| Results|| |
A total of 50 patients with resectable colorectal cancer were prospectively included in this study. These patients were randomly divided into two equal groups: the stained group and the unstained group.
In the stained group, surgical resection specimens were subjected to ex-vivo methylene blue injection into the feeding mesocolic artery.
In the unstained group, conventional pathological assessment was carried out.
Each group consisted of both sexes, male and female, of different ages.
Stained group (group 1)
In the stained group, there were 16 (64%) male and nine (36%) female patients, with a median age of 57 years (range: 26–81 years).
Unstained group (group 2)
In the unstained group, there were 11 (44%) male and 14 (56%) female patients, with a median age of 52 years (range: 28–80 years).
Differences between the two groups according to demographic data were statistically nonsignificant [Table 1].
|Table 1 Comparison between the two studied groups according to demographic data|
Click here to view
As regards tumor site (i.e., right colon, transverse, or left colon), no statistically significant difference was detected between the two groups [Table 2].
|Table 2 Comparison between the two studied groups according to tumor site|
Click here to view
As regards the surgical procedure, abdominal resection (i.e., right hemicolectomy) was performed in nine (36%) cases of the stained group and nine (36%) cases of the unstained group. However, extended right hemicolectomy was performed in seven (28%) cases of the stained group and four (16%) cases of the unstained group.
Left hemicolectomy was performed in two (8%) cases of the stained group and eight (32%) cases of the unstained group. However, anterior resection was performed in seven (28%) cases of the stained group and four (16%) cases of the unstained group. No statistically significant difference was detected between the two groups according to the type of operation [Table 3].
|Table 3 Comparison between the two studied groups according to the type of operations|
Click here to view
Methylene blue injection was successfully performed in all cases of the stained group, followed by final pathological staging. A statistically significant difference in pathological nodal stage (pN) was found between the two groups. In the stained group, 12 (48%) cases were finally staged as pN1 and nine (36%) cases were finally staged as pN2, whereas in the unstained group, nine (36%) cases were finally staged as pN1 and four (16%) cases were finally staged as pN2. However, there was a statistically nonsignificant difference in primary tumor stage (T stage) between the two groups [Table 4].
As regards the postoperative pathological stage, there was a statistically significant difference in pathological staging between the two groups. In the stained group, no cases (0%) were finally staged as stage I, four (16%) cases were staged as stage II, and 21 (84%) cases were staged as stage III. However, in the unstained group, three (12%) cases were staged as stage I, nine (36%) cases were staged as stage II, and 13 (52%) cases were staged as stage III [Table 5].
|Table 5 Comparison between the two studied groups according to pathological staging|
Click here to view
In the stained group, a total of 659 lymph nodes were identified with a mean of 26.36 ± 11.61, compared with a total number of 324 lymph nodes identified in the unstained group with a mean of 12.96 ± 4.25. The difference between the two groups was highly statistically significant (P = 0.001). At least 12 lymph nodes were identified in 24 (96%) of 25 cases of the stained group. However, in the unstained group, adequate nodal harvest was found in only 19 (76%) of 25 cases [Table 6].
|Table 6 Comparison between the two studied groups according to lymph node harvest|
Click here to view
Metastasis was found in 96 lymph nodes in 21 of 25 cases (84% of stained group cases) with a mean of 3.84 ± 2.87 and in 57 lymph nodes in 15 of 25 cases (60% of the unstained group cases) with a mean of 2.28 ± 3.13 [Table 6].
The difference between the two groups was statistically significant (P = 0.001).
The lymph nodes were measured and categorized by size. The difference in nodal recovery between the two groups was most pronounced in lymph nodes measuring less than or equal to 1 mm in diameter. The difference for the 1–3 mm categories was statistically highly significant (P = 0.001). However, for the lymph nodes in the more than 3 mm category the difference between the stained and unstained groups was statistically nonsignificant [Table 7].
|Table 7 Comparison between the two studied groups according to lymph node size|
Click here to view
| Discussion|| |
Although many published studies have tried to establish a minimal number of examined lymph nodes in colorectal cancer specimens, it is still not clear whether there is an absolute number that provides a gold standard under a routine pathological analysis .
However, the American Joint Committee on Cancer and the International Union Against Cancer recommend at least 12 lymph nodes to be examined for accurate colorectal cancer staging .
Different concepts have been developed to improve lymph node harvest and accuracy of staging in colorectal cancer. One group of techniques follows a strategy to heighten the sensitivity of metastasis detection in a single or in all collected lymph nodes. This group includes the sentinel lymph node technique, immunohistochemical analysis, and PCR .
A new and simple technique was described to stain lymph nodes in rectal cancer specimens using ex-vivo methylene blue injection into the inferior mesenteric artery, making them easier to detect by means of conventional pathological dissection. This technique was first described by Märkl and colleagues.
In our present study, lymph node recovery from randomly assigned 25 colorectal cancer specimens that underwent methylene blue injection into the feeding artery of the specimen (the stained group) was compared with that from an equal number of cases following conventional pathological examination (the unstained group) (total 50 cases) and the two groups were balanced as there was no statistically significant difference between them according to demographic, anatomical, and histological criteria.
In the present study, the process of methylene blue injection into the feeding mesocolic artery of the specimen was considered as a simple, easy, and safe method as it was successful in all cases of the stained group. Moreover, the availability and low cost of methylene blue dye and the fact that it possesses no hazardous or poisonous effects are the advantages. This coincides with the results of similar studies assessing this technique. Kerwel and colleagues described this technique to be simple, time-effective, and cost-effective and to be reproducible in other institutions, particularly where inadequate nodal harvests are problematic.
In our study, there was a major improvement in the median lymph node harvest in the stained group as compared with the unstained group (25 vs. 14, P = 0.001).
Moreover, mean lymph node harvest was higher in the stained group as compared with the unstained group (26.36 ± 11.61 vs. 12.96 ± 4.25, P = 0.001). In addition, in 96% of cases of the stained group, lymph node recovery was adequate according to the current American Joint Committee on Cancer and International Union Against Cancer guidelines, whereas in the unstained group only 76% of cases met the minimum requirement of 12 lymph nodes. This major improvement in nodal harvest following methylene blue staining of lymph nodes was similarly found by other published studies.
However, on comparing the results of our present study with other published results, it seems clear that nodal harvest after methylene blue lymph node staining in this study is the lowest among all with regard to the mean number for total lymph node harvested. However, our study has a better result compared with the study by Borowski et al. (2014) as regards the median (25 vs. 23). This indicates a more obvious need to improve lymph node harvests aiming at a more accurate colorectal cancer staging.
As regards the additional lymph nodes identified in the stained group, the increase was attributed to identifying more small lymph nodes. This difference in nodal recovery was most pronounced in lymph nodes measuring less than or equal to 3 mm in diameter (P = 0.001). Kerwel et al.  found similar results as the largest improvement was found in size groups between 1 and 4 mm causing a shift in size distribution toward smaller nodes.
The significance of the smaller lymph nodes is that studies have reported that 66–78% of metastatic lymph nodes are smaller than 5 mm .
Corresponding to the improvement in lymph node harvest in the stained group and the shift toward identifying smaller lymph nodes, a significant improvement was detected in the number of metastatic lymph nodes identified in the stained group as a mean metastatic lymph nodes of 3.84 ± 2.87 was found in the stained group compared with 2.28 ± 3.13 in the unstained group (P = 0.001). In contrast, in other published studies, methylene blue staining of lymph nodes did not lead to improvement in metastasis detection in rectal cancer .
For example, in the study by Kerwel and colleagues, no significant improvement was demonstrated in the detection of nodal metastasis as mean metastatic lymph nodes of 2 ± 3 and 2 ± 4 were found in the stained and unstained groups, respectively. This was attributed to the finding that nodal harvest in the unstained group was already very good and well above the reported averages obtained with conventional techniques .
In the present study, 84% of cases of the stained group were finally staged as stage III compared with 52% of cases of the unstained group.
To date, thorough examination of postoperative specimens in trial to obtain the maximum number of lymph nodes is the optimal method for accurate nodal staging .
Several authors claim that an insufficient number of identified and examined lymph nodes is one of the main causes of understaging in colorectal cancer. The stage migration theory is based on this concept .
| Conclusion|| |
This study demonstrates that ex-vivo methylene blue injection into the feeding mesocolic artery of the resected specimen for colorectal cancer is a simple, easy, and safe method that significantly improves lymph node harvesting in colorectal cancer leading to a more accurate colorectal cancer staging.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
El-Bolkainy TN, Sakr MA, Nouh AA, Ali El-Din NH. A comparative study of rectal and colonic carcinoma: demographic, pathological and TNM staging analysis. J Egypt Natl Canc Inst 2006; 18:258–263.
Mohammed AENS, Kandil MA, Asaad NY, Aiad HA, El Tahmoudy MA, Hemida AS. Immunohistochemical expression of Twist in colorectal carcinoma. Menoufia Med J 2015; 28:725–733. [Full text]
Wood WC, Skandalakis JE, Staley CA. Anatomic basis of tumor surgery. In: Lin E, editor. Colon and appendix
ed. Heidelberg, Germany: Springer-Verlag Berlin; 2010:378–399.
Compton CC. Colorectal carcinoma: diagnostic, prognostic, and molecular features. Mod Pathol 2003; 16:376–388.
Tepper JE, O'Connell MJ, Niedzwiecki D, Hollis D, Compton C, Benson AB, et al.
Impact of number of nodes retrieved on outcome in patients with rectal cancer. J Clin Oncol 2001; 19:157–163.
Johnson P, Malatjalian D, Porter G. Adequacy of nodal harvest in colorectal cancer: a consecutive cohort study. J Gastrointest Surg 2002; 6:883–890.
Wright FC, Law CH, Berry S, Smith AJ. Clinically important aspects of lymph node assessment in colon cancer. J Surg Oncol 2009; 99:248–255.
Märkl B, Kerwel TG, Jähnig HG, Oruzio D, Arnholdt HM, Scöler C, et al.
Methylene blue-assisted lymph node dissection in colon specimens. Am J Clin Pathol 2008; 130:913–919.
Braat AE, Oosterhuis WA, de Vries JE, Tollenaar R. Lymphatic staging in colorectal cancer: pathologic molecular and sentinel techniques. Dis Colon Rectum 2005; 48:371–383.
Märkl B, Kerwel TG, Wagner T, Anthuber M, Arnholdt HM. Methylene blue injection into the rectal artery as a simple method to improve lymph node harvest in rectal cancer. Mod Pathol 2007; 20:797–801.
Cianchi F, Palomba A, Boddi V, Messerini L, Pucciani F, Perigli G, et al.
Lymph node recovery from colorectal specimens: recommendation for a minimum number of lymph nodes to be examined. World J Surg 2002; 26:384–389.
Edge S, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC cancer staging manual handbook. From the AJCC Cancer Staging Manual. 7th
ed. New York, NY: Springer; 2010:143–151.
Tuech JJ, Pessaux P, di Fiore F. Sentinel node mapping in colon carcinoma: in-vivo
versus ex-vivo approach. Eur J Surg Oncol 2006; 32:158–161.
Kerwel TG, Spatz H, Anthuber M, Wünsch K, Arnholdt HM, Märkl B. Injecting methylene blue into the inferior mesenteric artery assures an adequate lymph node harvest and eliminates pathologist variability in nodal staging for rectal cancer. Dis Colon Rectum 2009; 52:935–941.
Schofield JB, Mounter NA, Mallett R, Haboubi NY. The importance of accurate pathological assessment of lymph node involvement in colorectal cancer. Colorectal Dis 2006; 8:460–470.
Chang GJ, Rodriguez-Bigas MA, Skibber JM, Moyer VA. Lymph node evaluation and survival after curative resection of colon cancer: systemic review. J Natl Cancer Inst 2007;99:433–441.
Törnroos A, Shabo I, Druvefors B, Arbman G, Olsson H. Postoperative intra-arterial methylene blue injection of colorectal cancer specimens increases the number of lymph nodes recovered. Histopathology 2011; 58:408–413.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]